Disulfide-Linked Reversible Terminators for NGS Read Length
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Solution Overview
Problem
Current Next Generation Sequencing (NGS) technologies face challenges with short read lengths due to reversible terminators leaving behind scars that impair DNA stability, hindering substrate recognition and primer extension, and existing solutions often require harsh conditions for cleavage or result in incomplete incorporation or removal of blocking groups.
Innovation Solution
Development of nucleotide analogs with a 3′-azidomethyl blocking group and a disulfanylalkoxycarbonylamino linked fluorophore, which can be cleaved using trialkylphosphine, allowing for simultaneous removal of the fluorophore and disulfide bond, leaving a minimal scar and enabling continued polymerase extension.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If reversible terminators are used in sequencing by synthesis, then accurate determination of incorporated base identity is achieved, but scars are left behind that impair DNA stability and hinder substrate recognition
Solution Approach 1:
The patent extracts and removes the problematic scar-forming components by using a cleavable blocking group that can be completely removed after serving its termination function. The blocking group is designed to be cleaved off along with the fluorophore, leaving no residual scar on the DNA strand, thus resolving the contradiction between achieving accurate base identification and maintaining DNA stability.
Solution Approach 2:
The blocking group and fluorophore are designed as temporary, discardable components that fulfill their detection function and then are completely removed. The cleavable linker enables complete removal of these components without leaving residual structures, allowing the DNA strand to recover its natural state and stability while still enabling accurate sequencing.
2Reliability
If existing cleavage methods are used to remove blocking groups, then terminators are cleaved, but harsh conditions are required or incomplete removal occurs
Solution Approach 1:
The patent changes the chemical parameters of the blocking group by incorporating a disulfide bond that is sensitive to mild reducing conditions. This allows the blocking group to be removed under gentle conditions (mild reducing agents) rather than harsh conditions, while ensuring complete removal. The fluorophore is also designed with a disulfide linker for simultaneous complete removal under the same mild conditions.
Solution Approach 2:
The disulfide bond acts as an intermediary that enables complete and clean removal of both the blocking group and fluorophore under mild conditions. The disulfide linker provides a controlled cleavage mechanism that ensures complete removal without requiring harsh conditions, resolving the contradiction between reliable complete removal and avoidance of harmful cleavage conditions.
3Ease of operation
If blocking groups are placed at the 3′-OH position to impede polymerase, then chain termination is achieved, but DNA stability is compromised due to scar formation
Solution Approach 1:
The patent extracts the blocking function from a permanent modification and makes it temporary and removable. The blocking group is designed to be completely removed after serving its chain-termination function, leaving no scar on the DNA strand. This resolves the contradiction by maintaining ease of operation (chain termination control) while preserving DNA stability through complete removal of the blocking group.
4Measurement precision
If detectable labels are attached through cleavable linkers, then base identity detection is enabled, but free thiol groups may be generated after cleavage
Solution Approach 1:
The patent converts the potential harm of free thiol group generation into a benefit by designing the disulfide linker to undergo intramolecular cyclization after cleavage. The released thiol group immediately cyclizes to form a stable five-membered ring, eliminating the harmful free thiol while maintaining the benefit of complete and clean label removal. This resolves the contradiction between enabling detectable label attachment and preventing harmful free thiol generation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for accurate determination of incorporated nucleotides with minimal disruption to the DNA strand, enabling longer read lengths and improved sequencing accuracy without damaging the DNA or polymerase, under mild reaction conditions.
Implementation Method 1
The cleavable linker comprises a disulfide bond which can be cleaved by a chemical reagent at the same time when the same chemical reagent cleaves the cleavable chemical group on the 3′ hydroxyl of the nucleotide sugar moiety
Data Source
AI summary
The present disclosure provides methods of sequencing polynucleotides and compounds, compositions useful for sequencing of polynucleotides. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base that is attached to a label through a cleavable linker comprising a disulfide bond. In addition, both the disulfide bond and the cleavable chemical group are cleavable by a chemical reagent. Furthermore, after the disulfide bond is cleaved by the chemical reagent, there is no free thiol group linked to the base of the nucleotides according to the fragmentation reaction shown below as an example. Example compounds according to the present disclosure are shown as Formula (I):wherein w is 1-5; X is O, S, or BH3; B is a nucleotide base or an analog thereof, L1-3 are linkers; and D1 is a label.